Mycobacterium kansasii is a slowly growing photochromogenic bacterium that causes chronic pulmonary disease resembling tuberculosis (Wayne. L. G. and G. P. Kubica, 1986, "The Mycobacteria," pp. 1435-1457, in Sneath et al., eds., BERGEY'S MANUAL OF SYSTEMIC BACTERIOLOGY, Vol. 2, Williams and Wilkins, Baltimore). Among mycobacteria other than M. tuberculosis and M. avium complex strains, M. kansasii is one of the most frequently isolated species.
Disseminated infections caused by non-tuberculosis mycobacteria such as M. kansasii have become an increasing public health concern as the number of AIDS infected individuals increases. M kansasii is currently the second most common nontuberculosis mycobacterium causing disseminated disease in HIV-infected patients (after the M. avium complex).
Classical methods for identification of mycobacteria involve various biochemical techniques, acid fast staining, cell morpholiogy and HPLC analysis. M. kansasii cells are moderately long to long rods. Colonies range from flat to raised and smooth to rough colony types. M. kansasii colonies are typically nonpigmented when grown in the dark and turn yellow following exposure to light (photochromogenic). Biochemical tests include positive nitrate reduction, tween and urea hydrolysis, catalase activity and niacin production. It can take several months to speciate a mycobacteria isolate using these identification methods.
Certain subspecies of M. kansasii are atypical. See for example Ross et al., J. Clin. Microbiol. 30:2930-2933 (1992). These atypical subspecies have variations in their 23S rRNA sequence, and therefore are not necessarily detectable with probes directed to 23S rRNA derived from the typical strains of M. kansasii. However, these atypical strains have been implicated as causative agents in infections, and it is therefore important to be able to identify the atypical strains as M. kansasii. Therefore, the term M. kansasii as used herein refers to both typical and atypical strains of the organism.
It is therefore an object of the present invention to provide nucleic acid hybridization probes for the rapid and specific detection of M. kansasii in test samples and particularly in human clinical specimens. Further, it is an object of the present invention to provide probes capable of detecting formerly undetectable subspecies of M. kansasii.
As used herein, the term "test sample" is intended to mean any sample suspected of containing the intended target nucleic acid, and includes but is not limited to: biological samples, body fluids or exudate such as urine, blood, milk, cerebrospinal fluid, sputum, saliva, stool, lung aspirates, throat or genital swabs, clinical specimens containing one or more of the foregoing, environmental samples, food samples and laboratory samples.
Nucleic acid hybridization is the process by which two nucleic acid strands having completely or partially complementary nucleotide sequences come together under predetermined reaction conditions to form a stable, double-stranded hybrid with specific hydrogen bonds. Either nucleic acid strand may be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA); thus hybridization can involve RNA:RNA hybrids, DNA:DNA hybrids, or RNA:DNA hybrids.
Thus, as used in this application, the term "hybridization" refers to the ability of two completely or partly complementary single nucleic acid strands to come together in an antiparallel orientation to form a stable structure having a double-stranded region. The two constituent strands of this double-stranded structure, sometimes called a hybrid, are held together with hydrogen bonds. Although these hydrogen bonds most commonly form between nucleotides containing the bases adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G), base pairing can form between bases which are not members of these "canonical" pairs. Non-canonical base pairing is well-known in the art. See e.g., The Biochemistry of the Nucleic Acids (Adams et al., eds., 1992).
Nucleic acid hybridization is a common method for detecting and quantitating target nucleic acids having specific nucleotide sequences. Such methods are useful for identifying and classifying organisms, diagnosing infectious diseases and genetic abnormalities, testing food and drugs, and identifying criminal suspects, among numerous other goals. Typically, nucleic acid hybridization assays use a labeled oligonucleotide hybridization assay probe having a nucleic acid sequence complementary to the target sequence. Such labels are well known in the art, and may include radioactive isotopes, enzymes, or fluorescent, luminescent, or chemiluminescent groups; the Applicants prefer the use of chemiluminescent acridinium esters as labels. See Arnold et al. U.S. Pat. No. 5,185,439, which enjoys common ownership with the present application and is incorporated by reference herein. The probe is mixed with a sample suspected of containing a nucleic acid having the target sequence under hybridization conditions suitable for allowing annealing of the two strands by hydrogen bonding in the region of complementarity. The probe then hybridizes to the target nucleic acid present in the sample. The resulting hybrid duplex may be detected by various techniques well known in the art, such as hydroxyapatite adsorption. Also included among these techniques are those that involve selectively degrading the label present on unhybridized probe and then measuring the amount of label associated with the remaining hybridized probe, as disclosed in Arnold et al., U.S. Pat. No. 5,283,174, which enjoys common ownership with the present application and is incorporated by reference herein. This latter technique, called the hybridization protection assay (HPA), is presently preferred by the Applicants.
Often a test sample will not contain a great enough number of nucleic acid molecules to permit direct detection or quantification by nucleic acid hybridization due to the sensitivity limits of the particular label used. In such a case, the amount of detectable target nucleotide sequence is increased before nucleic acid hybridization is used to identify its presence or amount in the test sample. This procedure is termed nucleic acid amplification, and the method of increasing the amount of the target nucleic acid is referred to as amplifying the target nucleic acid or target nucleotide sequence.
Amplification methods involve the use of at least one nucleic acid strand containing a target nucleotide sequence as a template in a nucleic acid polymerizing reaction to produce a complementary second strand containing the target nucleotide sequence. Amplification can be performed on both the sense and anti-sense strands of a duplex nucleic acid molecule containing the target nucleotide sequence. By repeating this process, using the product nucleic acids as templates in subsequent cycles, the number of nucleic acid molecules having the target nucleotide sequence increases rapidly.
A number of amplification methods have been described; among these are various embodiments of the polymerase chain reaction (PCR), (see e.g., Mullis et al., U.S. Pat. No. 4,683,195), and methods which utilize in vitro transcription (RNA synthesis) in one or more step of the procedure, (see e.g., Murakawa et al., DNA 7:287-295, Burg et al. PCT Publication No. W089/1050, Gingeras et al., PCT Publication No. WO088/10315, Gingeras et al. European Patent No. EP0373960, McDonough, et al., PCT Publication No. WO 94/03472, Kacian, et al., PCT Publication No. WO 93/22461, and Dattagupta, et al. (filed in the United States Mar. 16, 1994, U.S. patent application Ser. No. 08/215,081). The disclosure of these references are incorporated by reference herein; the last two of these references enjoy common ownership with the present application.
A hybridization assay probe is used to detect, indicate and/or quantify the presence of the intended target nucleic acid; such a probe is usually labeled with a radioactive or luminescent atom or a detectable chemical group, such as a chemiluminescent moiety. Applicants prefer using acridinium ester derivatives as a labeling reagent. However, the presence of the intended target nucleic acid can also be detected without the use of a labeled probe. For example, hybrids formed between the probe and the target nucleic acid can be isolated using hydroxyapitite or gel filtration, or can be visualized by using non-denaturing gel electrophoresis. Sometimes the intended target nucleic acid will include any of a population of different nucleic acid molecules with nucleotide sequences usually derived from a biological source. By way of example only, and not of limitation, the target nucleotide sequence may be shared by the nucleic acids of a genus of organisms (but not by organisms outside the genus), the detection of any of which is desired. Alternatively, the target nucleotide sequence may be unique to a species of organism or to a strain of that species.
Not all probes are necessarily intended to be detectable. Some hybridization probes, termed "helper oligonucleotides" or "helper probes," are designed to facilitate the ability of a separate assay probe to bind to its target nucleotide sequence. Although not wishing to be bound by theory, Applicants believe helper probes facilitate binding of the assay probe by locally decreasing the amount of intramolecular hydrogen-bonding in the target nucleic acid, thus making the target nucleotide sequence more available for specific hybridization with the labeled probe. Depending on the location of the labeled probe's binding site and the secondary structure of the target nucleic acid, helper probes may be directed to nucleotide sequence regions proximal to the labeled probe's binding site, or directed to regions distal from the binding site which nevertheless affect probe binding. Helper probes are described in Hogan et al., U.S. Pat. No. 5,030,557 which enjoys common ownership with the current application, and which is incorporated by reference herein.
Descriptions of the use of nucleic acid hybridization to detect the presence of particular nucleic acid sequences are given in Kohne, U.S. Pat. No. 4,851,330 and in Hogan et al., International Patent Application No. PCT/US87/03009; both of these references enjoy common ownership with the present application, and are incorporated by reference herein. Hogan describes methods for determining the presence of a non-viral organism or a group of non-viral organisms in a sample (e.g., sputum, urine, blood and tissue sections, food, soil and water) using nucleic acid hybridization techniques.
Hogan, supra, also describes a number of hybridization probes which specifically detect only targeted ribosomal RNA (rRNA) nucleotide sequences belonging to a specific organism or group of organisms.